Antibody proteins play a key role in the identification and/or destruction of agents within an organism. Antibodies have the ability to specifically bind to certain antigens while not binding to other antigens, despite the antigens having similar structures. However, because of the high specificity, it is necessary for most organisms to have the capacity to make thousands of different antibodies, each antibody specific for a particular antigen.
The human body employs several different immunoglobulin classes of antibodies, depending upon the role the antibody plays in the immune response. Each immunoglobulin class may have thousands of particular variations depending upon the antigen to which the antibody responds. Identified classes include IgG, IgA, IgM, IgD, and IgE. IgG is the most prevalent antibody class found in human serum. A structure of an IgG protein is shown in FIG. 1. As with all IgGs, the structure in FIG. 1 has two binding domains (Fab) and a central, non-binding domain (Fc).
The specific affinity of an IgG antibody for an antigen is a result of amino acid variations in the antigen binding sites at the ends of the Fab fragments. The specific affinity is often described as a “lock and key” mechanism, as is illustrated in FIG. 2. Because only particular antigens have a structure matching the antigen binding sites of a particular antibody, that antibody will bind only that antigen, and no others. For example, an antibody against human serum albumin (HSA) will bind HSA in human serum, while ignoring the thousands of other proteins in the serum.
Antibodies can be created de novo against specific antigens using monoclonal antibody techniques in mice, for example. Using these techniques, researchers can obtain new antibodies against known antigens, which can then be used for research and assays, such as ELISA (defined below) and Western blotting. Additionally, thousands of different antibodies are readily available from a number of suppliers, such as Sigma Aldrich of St. Louis, Mo. A comprehensive list of antibody suppliers is available from Linscott's directory (http://www.linscottsdirectory.com/search/antibodies). Additionally, it is possible to raise antibodies against non-biological agents, such as chemical compounds derived from petroleum. For example, antibodies may be raised against melamine, which has been linked to contaminated infant formulas.
ELISA (Enzyme-Linked Immuno-Sorbent Assay) incorporates antibodies to detect specific chemical or biological species. In one method of ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody (linked to an enzyme) is washed over the surface so that it can bind to the antigen. The surface is then washed with a buffer to remove any unbound antibodies.
By adding a chemical species that is converted to a fluorescent or colorimetric molecule by the enzyme, it is possible to infer the presence of antigens for which the known antibodies are specific. That is, bound antibody-enzyme complexes will fluoresce in the presence of light of the appropriate wavelength or otherwise be spectroscopically detectable. However, if no antibodies have bound, there will be no fluorescence or colorimetric change. By measuring the intensity of the fluorescence or color produced it is also possible to infer a relative amount of antigen present in the sample.
ELISA suffers from a number of limitations. It is time- and labor-intensive, requiring several preparation and wash steps, and quantitative measurements require the use of expensive light sources and detection equipment (e.g., a fluorimeter or spectrophotometer). However, the sensitivity and specificity of ELISA offsets these limitations. Because of the sensitivity and specificity, ELISA is commonly used to detect the presence of viruses, such as HIV, in human serum. ELISA is also commonly used in the food processing industry to screen for allergens, such as peanut or egg proteins or other contaminants such as chemicals or microorganisms. It is also known to employ parallel ELISA testing with multiple antibody solutions, e.g., using a 96 well microtiter plate.
The specificity and sensitivity of ELISAs are the consequence of using antibodies that have been chosen because of their specificity to the antigens that are being screened. Nonetheless, a lower-cost antibody-based sensor may allow for greater utilization of antibody detection. For example, massive parallel antibody screening would be very useful in the fields of genomics and proteomics.
Furthermore, if antibody-based sensors could be produced that were both portable and stable over long periods of time, such sensors could be deployed by homeland security agencies, allowing the agencies to quickly detect and characterize pathogens in the event of a suspected chemical or biological weapons attack.